Bottom Line:
Pharmacological inhibition of MMP activity suppressed the expression of Na(v)1.7/1.8 channels in the crushed nerves.Collectively, our data established an essential role of the MMP-9/TIMP-1 axis in guiding the mSC differentiation and the molecular assembly of myelin domains in the course of the nerve repair process.Our findings of the MMP-dependent regulation of Na(v) channels, which we document here for the first time, provide a basis for therapeutic intervention in sensorimotor pathologies and pain.

Affiliation: Department of Anesthesiology, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT

Background: Myelinating Schwann cells (mSCs) form myelin in the peripheral nervous system. Because of the works by us and others, matrix metalloproteinase-9 (MMP-9) has recently emerged as an essential component of the Schwann cell signaling network during sciatic nerve regeneration.

Methodology/principal findings: In the present study, using the genome-wide transcriptional profiling of normal and injured sciatic nerves in mice followed by extensive bioinformatics analyses of the data, we determined that an endogenous, specific MMP-9 inhibitor [tissue inhibitor of metalloproteinases (TIMP)-1] was a top up-regulated gene in the injured nerve. MMP-9 capture followed by gelatin zymography and Western blotting of the isolated samples revealed the presence of the MMP-9/TIMP-1 heterodimers and the activated MMP-9 enzyme in the injured nerve within the first 24 h post-injury. MMP-9 and TIMP-1 co-localized in mSCs. Knockout of the MMP-9 gene in mice resulted in elevated numbers of de-differentiated/immature mSCs in the damaged nerve. Our comparative studies using MMP-9 knockout and wild-type mice documented an aberrantly enhanced proliferative activity and, accordingly, an increased number of post-mitotic Schwann cells, short internodes and additional nodal abnormalities in remyelinated nerves of MMP-9 knockout mice. These data imply that during the first days post-injury MMP-9 exhibits a functionally important anti-mitogenic activity in the wild-type mice. Pharmacological inhibition of MMP activity suppressed the expression of Na(v)1.7/1.8 channels in the crushed nerves.

Conclusion/significance: Collectively, our data established an essential role of the MMP-9/TIMP-1 axis in guiding the mSC differentiation and the molecular assembly of myelin domains in the course of the nerve repair process. Our findings of the MMP-dependent regulation of Na(v) channels, which we document here for the first time, provide a basis for therapeutic intervention in sensorimotor pathologies and pain.

pone-0033664-g001: Heatmap of the genome-wide transcriptional profiling data of the murine sciatic nerve and the corresponding L4/5 DRG from the same animals.Red and blue correspond to the high and the low expression levels, respectively. Color map inset shows the signal intensity scale. Only the genes with the B∶A ratio >4 are shown in a descending order. A, sham nerve; B, nerve day 1 post-transection; C, nerve day 5 post-transection; D, DRG corresponding to the sham-operated nerve; E, DRG corresponding to day 5 post-transection. The green dots point to TIMP-1, TNC, NGAL, CD44, and MMP-9 induced in the injured nerve, and MMP-13 in the DRG. DRG, dorsal root ganglia.

Mentions:
To identify the genes the expression of which was affected in the sciatic nerve as a result of the injury, we performed genome-wide transcriptional profiling of the transected and sham-operated mouse sciatic nerves and the associated lumbar (L)4/5 dorsal root ganglia (DRG) at days 1 and 5 post-transection. Because SCs de-differentiate immediately distal and immediately proximal to injury [2], [17], both segments of the transected nerve were pooled together for analyses and identified as the “injured” sample. We identified that the expression of multiple genes was affected in the injured nerve compared with a sham control. The listing and the heatmap of the 50 top up-regulated genes are shown in Table 1, and Fig. 1 and 2, respectively. The gene expression data have been deposited to GEO database (accession # GSE33454). In agreement with the earlier reports [45], [46], [47], [48], the injury up-regulated the expression of the genes coding for arginase I (an enzyme involved in polyamine synthesis [4]), calcium-binding S100A8/A9, chemokine cc- and cxc-motif ligands (e.g., ccl2-4, ccl7, cxcl1, cxcl10 and cxcl14), cytokine ligands and receptors (e.g., interleukins il1b, il1r2 and il7r) and additionally, toll-like receptors (e.g., tlr1, tlr7, tlr2, tlr6 and tlr13). Furthermore, the injury caused a multi-fold up-regulation of the genes that are directly linked to proteolysis, cell adhesion, cell signaling, and maintenance of the extracellular matrix, including TIMP-1 (the top 6th up-regulated gene in the system), tenascin C (TNC) that is important in the immune response to tissue damage [42], [48], [49] and neutrophil gelatinase-associated lipocalin-2 (NGAL)/lipocalin-2 (LCN2), known to directly interact with MMP-9 [50], [51].

pone-0033664-g001: Heatmap of the genome-wide transcriptional profiling data of the murine sciatic nerve and the corresponding L4/5 DRG from the same animals.Red and blue correspond to the high and the low expression levels, respectively. Color map inset shows the signal intensity scale. Only the genes with the B∶A ratio >4 are shown in a descending order. A, sham nerve; B, nerve day 1 post-transection; C, nerve day 5 post-transection; D, DRG corresponding to the sham-operated nerve; E, DRG corresponding to day 5 post-transection. The green dots point to TIMP-1, TNC, NGAL, CD44, and MMP-9 induced in the injured nerve, and MMP-13 in the DRG. DRG, dorsal root ganglia.

Mentions:
To identify the genes the expression of which was affected in the sciatic nerve as a result of the injury, we performed genome-wide transcriptional profiling of the transected and sham-operated mouse sciatic nerves and the associated lumbar (L)4/5 dorsal root ganglia (DRG) at days 1 and 5 post-transection. Because SCs de-differentiate immediately distal and immediately proximal to injury [2], [17], both segments of the transected nerve were pooled together for analyses and identified as the “injured” sample. We identified that the expression of multiple genes was affected in the injured nerve compared with a sham control. The listing and the heatmap of the 50 top up-regulated genes are shown in Table 1, and Fig. 1 and 2, respectively. The gene expression data have been deposited to GEO database (accession # GSE33454). In agreement with the earlier reports [45], [46], [47], [48], the injury up-regulated the expression of the genes coding for arginase I (an enzyme involved in polyamine synthesis [4]), calcium-binding S100A8/A9, chemokine cc- and cxc-motif ligands (e.g., ccl2-4, ccl7, cxcl1, cxcl10 and cxcl14), cytokine ligands and receptors (e.g., interleukins il1b, il1r2 and il7r) and additionally, toll-like receptors (e.g., tlr1, tlr7, tlr2, tlr6 and tlr13). Furthermore, the injury caused a multi-fold up-regulation of the genes that are directly linked to proteolysis, cell adhesion, cell signaling, and maintenance of the extracellular matrix, including TIMP-1 (the top 6th up-regulated gene in the system), tenascin C (TNC) that is important in the immune response to tissue damage [42], [48], [49] and neutrophil gelatinase-associated lipocalin-2 (NGAL)/lipocalin-2 (LCN2), known to directly interact with MMP-9 [50], [51].

Bottom Line:
Pharmacological inhibition of MMP activity suppressed the expression of Na(v)1.7/1.8 channels in the crushed nerves.Collectively, our data established an essential role of the MMP-9/TIMP-1 axis in guiding the mSC differentiation and the molecular assembly of myelin domains in the course of the nerve repair process.Our findings of the MMP-dependent regulation of Na(v) channels, which we document here for the first time, provide a basis for therapeutic intervention in sensorimotor pathologies and pain.

Affiliation:
Department of Anesthesiology, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT

Background: Myelinating Schwann cells (mSCs) form myelin in the peripheral nervous system. Because of the works by us and others, matrix metalloproteinase-9 (MMP-9) has recently emerged as an essential component of the Schwann cell signaling network during sciatic nerve regeneration.

Methodology/principal findings: In the present study, using the genome-wide transcriptional profiling of normal and injured sciatic nerves in mice followed by extensive bioinformatics analyses of the data, we determined that an endogenous, specific MMP-9 inhibitor [tissue inhibitor of metalloproteinases (TIMP)-1] was a top up-regulated gene in the injured nerve. MMP-9 capture followed by gelatin zymography and Western blotting of the isolated samples revealed the presence of the MMP-9/TIMP-1 heterodimers and the activated MMP-9 enzyme in the injured nerve within the first 24 h post-injury. MMP-9 and TIMP-1 co-localized in mSCs. Knockout of the MMP-9 gene in mice resulted in elevated numbers of de-differentiated/immature mSCs in the damaged nerve. Our comparative studies using MMP-9 knockout and wild-type mice documented an aberrantly enhanced proliferative activity and, accordingly, an increased number of post-mitotic Schwann cells, short internodes and additional nodal abnormalities in remyelinated nerves of MMP-9 knockout mice. These data imply that during the first days post-injury MMP-9 exhibits a functionally important anti-mitogenic activity in the wild-type mice. Pharmacological inhibition of MMP activity suppressed the expression of Na(v)1.7/1.8 channels in the crushed nerves.

Conclusion/significance: Collectively, our data established an essential role of the MMP-9/TIMP-1 axis in guiding the mSC differentiation and the molecular assembly of myelin domains in the course of the nerve repair process. Our findings of the MMP-dependent regulation of Na(v) channels, which we document here for the first time, provide a basis for therapeutic intervention in sensorimotor pathologies and pain.